This invention relates to methods and apparatus for producing electrophoretic gels which require polymerization of material during their formation.
The analysis of complex biochemical systems is dependent on technology which allows individual molecules to be separated. Standard techniques exist which allow protein, DNA, and RNA molecules to be separated and/or purified on the basis of their size, electrical charge, physical structure or a combination thereof. Most of these techniques employ at least one type of gel electrophoresis. In gel electrophoresis, the molecules to be separated are subjected to an electric field which causes them to enter a gel of a known pore size, charge and pH. Depending on the conditions, different molecules will be either retarded or accelerated as they pass through the gel, and hence separated.
One of the most common materials used for electrophoresis is polyacrylamide. A polyacrylamide gel is formed by the chemical cross-linking of a monomer, acrylamide, with a co-monomer, such as N,N'-methylenebisacrylamide (BIS), N,N'-bisacrylylcystamine (BAC), N,N'-(1,2-dihydroxyethylene) bisacrylamide (DHEBA), or N,N'-diallyltartardiamide (DATD). The ratio, as well as the concentration of these monomers determines the final pore size, and hence sieving properties of the gel. Chemical cross-linking, or polymerization, of the gel is effected by the formation of free radicals by an initiator, such as ammonium persulfate (APS) or riboflavin. Polymerization is made even more efficient by the addition of an accelerator such as N,N,N',N'-tetramethylethylenediamine (TEMED) or 3-dimethylaminoproprionitrile (DMAPN).
Non-acrylamide chemically cross-linked matrices have recently come to market. See e.g., AT Biochem, Inc. catalog. These are prepared in a manner similar to polyacrylamide gels.
Generally, an electrophoretic gel is prepared in the following manner. While wearing gloves, and typically in a fume hood, the user prepares the monomer and co-monomer as concentrated aqueous stock solutions which are stored at 4.degree. C. until use. An initiator such as APS is prepared on the day of use as an aqueous solution and stored at 4.degree. C. Accelerators such as TEMED are provided as liquids. Other components of the gel depend on the type of molecules to be separated. For example, for one-dimensional separation of proteins, water, Tris(hydroxymethyl)methylamine (TRIS) and sodium dodecyl sulfate (SDS) are usually prepared as aqueous stock solutions. These four stock solutions are then mixed to prepare a final liquid which is then poured into a suitable apparatus and allowed to polymerize to form a polyacrylamide gel.